Platelets are small, irregularly-shaped a-nuclear cells that play a fundamental role in hemostasis and healing. Platelets contain a complete array of pre-synthesized proteins, among which are signaling proteins, cytoskeletal proteins, membrane proteins and regulatory proteins. They are involved in key stages of tissue regeneration and healing processes at the site of injury, mainly due to the content of platelet granules comprising a multitude of bioactive molecules including growth factors (GFs), cytokines and chemokines. Platelet GFs such as platelet-derived growth factor (PDGF), transforming growth factor (TGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and others are key players in all the following phases of the wound healing cascade: inflammatory, proliferative and remodeling phase.
Studies have shown that platelet derived GFs stimulate angiogenesis, mitogenesis, cell proliferation, neutrophils and macrophages, collagen synthesis, wound contraction, extracellular matrix synthesis, epithelialization and chemotaxis.
Platelets are routinely used by transfusion e.g. to improve hemostasis. Recently, platelets are increasingly used in the form of Platelet Rich Plasma (PRP), also referred to as PRP gel, platelet gel, PRP-clot etc. Typically, PRP is an ex vivo preparation consisting of autologous platelets concentrated in a limited volume of plasma (Lacci K M, Dardik A. Platelet-rich plasma: support for its use in wound healing. Yale J Biol Med. 2010 March; 83(1):1-9).
For topical application, PRP is usually activated by the addition of thrombin and/or CaCl2 resulting in the formation of fibrin gel by the interaction between thrombin (endogenous or exogenous) and fibrinogen. Upon activation, the platelets undergo active degranulation and release various mediators including GFs (Lacci K M, Dardik A, 2010). The use of PRP for injection currently comprises a small but rapidly growing segment of the market. The rationale for using PRP in soft and hard tissue augmentation is its potential to enhance tissue regeneration in non-healing injuries, accelerate wound maturity, vascularization and epithelialization, decrease scar formation, and reduce post operative complications and morbidity (Lacci K M, Dardik A, 2010).
Studies using activated PRP together with various cell types have shown that factors e.g. growth factors released from PRP can induce cell proliferation [(e.g. Kanno et al. Platelet-rich plasma enhances human osteoblast-like cell proliferation and differentiation. J Oral Maxillofac Surg. 2005 March; 63(3):362-9; Bertrand-Duchesne et al. Epidermal growth factor released from platelet-rich plasma promotes endothelial cell proliferation in vitro. J Periodontal Res. 2010 February; 45(1):87-93; Kakudo et al. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts. Plast Reconstr Surg. 2008 November; 122(5):1352-60), modulate the angiogenic capability of human endothelial cells (Sulpice et al. Cross-talk between the VEGF-A and HGF signalling pathways in endothelial cells. Biol Cell. 2009 September; 101(9):525-39; Rughetti et al. Platelet gel-released supernatant modulates the angiogenic capability of human endothelial cells. Blood Transfus. 2008 January; 6(1):12-7), and induce osteo-inductive properties (Intini G. The use of platelet-rich plasma in bone reconstruction therapy. Biomaterials. 2009 October; 30(28):4956-66)]. Moreover, activated PRP was found to support in vitro cell growth and maintained viability of a number of target cells including myelomas, hybridomas, hepatocytes, fibroblasts and epithelial cells, at a level comparable or superior to the level supported by fetal bovine serum (Johansson et al. Platelet lysate: a replacement for fetal bovine serum in animal cell culture? Cytotechnology. 2003 July; 42(2):67-74).
PRP and released growth factors are currently used in various surgical tissue regeneration procedures, predominantly in orthopedic and dental surgery (Nurden et al. Platelets and wound healing. Front Biosci. 2008 May 1; 13:3532-48). In orthopedic surgery PRP is used mainly for knee arthroplasty, lumbar spinal fusion, and in intervertebral disc degeneration (reviewed in Nurden et al, 2008). Dentistry and maxillofacial surgery PRP applications include mainly consolidation of titanium implants, maxillary sinus augmentation and bone remodeling (reviewed in Nurden et al, 2008). PRP is also increasingly used for tendon and ligament repair, facial plastic and reconstructive surgery, chronic skin wound healing, ophthalmology, facial nerve regeneration, as well as in cardiac and bariatric surgery (reviewed in Nurden et al, 2008).
However, a major disadvantage of the current use of autologous PRP and released factors resides in the lack of standardization. Of note, different manual, semi-automated and fully-automated systems for preparation of PRP are commercially available that differ in parameters such as preparation time, platelet yield and collection efficiency (Mazzucco et al. Not every PRP-gel is born equal. Evaluation of growth factor availability for tissues through four PRP-gel preparations: Fibrinet, RegenPRP-Kit, Plateltex and one manual procedure. Vox Sang. 2009 August; 97(2):110-8).
Another important variable is the technique used for platelet activation [autologous, heterologous or recombinant thrombin, calcium chloride or batroxobin (Rozman P, Bolta Z. Use of platelet growth factors in treating wounds and soft-tissue injuries. Acta Dermatovenerol Alp Panonica Adriat. 2007 December; 16(4):156-65)], which can affect the efficiency of granule release and the amount of secreted GFs (Rozman P, Bolta Z, 2007). Moreover, since platelets are very sensitive to mechanical stress and changes in the surrounding environment, they may be activated and GFs may be released during processing, prior to the intended activation step (Mazzucco et al, 2009). This uncontrolled activation may further increase the variability in the composition of the final product when using different PRP preparation systems. Additionally, a major inherent weakness of autologous PRP preparation is that the platelets GFs content varies among individuals, and therefore may lead to sub-optimal results. Finally, the financial burden of dedicated machinery, disposable PRP processing kits, and the need for trained personnel, should be taken into consideration when working with autologous PRP.
A recent publication (Su et al. “A virally inactivated functional growth factor preparation from human platelet concentrates”. Vox Sang. 2009 August; 97(2):119-128) discloses the preparation of a clottable functional growth factor extract derived from pooled aphaeresis platelet donations. However, the disclosed clottable preparation has the disadvantage that it includes only one viral inactivation step, i.e. solvent detergent (S/D) viral inactivation which is effective particularly against enveloped viruses, but not against non-enveloped viruses. The publication indicates the possibility of applying nanofiltration as a second viral inactivation step. This preparation also contains plasma and leukocyte protein impurities. The step of S/D removal by hydrophobic interaction chromatography (HIC) largely reduces the PDGF in the preparation. Furthermore, the clotability potential of the preparation may limit its use to local application or prevent its systemic use.
Burnouf et al. (“A novel virally inactivated human platelet lysate preparation rich in TGF-beta, EGF and IGF, and depleted of PDGF and VEGF”. Biotechnol Appl Biochem. 2010 Aug. 6; 56(4):151-60) discloses an S/D treated platelet lysate with a standardized content of TGF-beta, EGF and IGF and depleted of PDGF and VEGF. The publication discloses a method for preparation of this lysate evading removal of SD by hydrophobic interaction chromatography.
There is a need of a viral-safe platelet extract preparation obtained from multiple donors comprising a mixture of proteins having growth factor and/or trophic factor activity.